Influence of oxidative stress and metabolic adaptation on PON1 activity and MDA level in transition dairy cows.

Serum PON1 is a HDL-associated enzyme that protects lipoproteins, both LDL and HDL, against oxidation and it is considered as an antioxidative/anti-inflammatory component of HDL. Dairy cows are highly susceptible to oxidative stress which commonly occurs in late pregnancy and early lactation. During the transition period, increased production of reactive oxygen species is associated to processes of metabolic adaptation to a low-energy balance. We investigated serum paraoxonase-1 (PON1) activity and malondialdehyde (MDA) concentration to assess the antioxidative/prooxidative status during pregnancy and the postpartum period. In order to evaluate metabolic homeostasis, common metabolic parameters (glucose, triglyceride, total cholesterol, HDL-C and albumin concentrations) were determined as well. A significantly lower PON1 activity was found in late pregnancy and early postpartum (P<0.05) compared to the first and the second trimester of pregnancy and the mid-lactation. MDA level was significantly higher (P<0.05) in the dry period compared to pregnant lactating and postpartum cows. Serum glucose concentration (P<0.001) was lower in the early and late puerperium indicating low-energy balance in the early lactation. Serum triglyceride and albumin concentrations were lower in late puerperium (P<0.001), while total cholesterol and HDL-C were lower during the dry period (P<0.05) as well as in early postpartum (P<0.001). Significant correlations of PON1 activity with glucose (P<0.05), albumin (P<0.05), total cholesterol (P<0.001) and HDL-C (P<0.001) were also found. The observed lower serum PON1 activity and higher MDA level in late pregnancy and early postpartum could indicate a prooxidants/antioxidants imbalance influenced by reproductive stress and metabolic adaptation in the transition period of dairy cows.

Glycine Substitution Reduces Antimicrobial Activity and Helical Stretch of diPGLa-H in Lipid Micelles.

With the rise in antibiotic resistance, antimicrobial peptides (AMPs) show promise for therapeutic development, but higher specificity is required. PGLa-H is a naturally occurring decapeptide, reported to have moderate antibacterial activity and low hemolytic activity, with its sequence being identical to that of the C-terminal fragment of highly selective AMP, PGLa. DiPGLa-H, a sequential tandem repeat of PGLa-H, and Kiadin, an analogue with a Val to Gly substitution at position 15, display improved in vitro bactericidal activity against both Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells. Despite Gly being a more flexible residue, NMR structural studies showed little difference in structure and dynamics between the two peptides for the first 14 residues, with somewhat greater flexibility in the C-terminus of Kiadin resulting in a tighter structure of the peptide in the presence of sodium dodecyl sulfate micelles. AMPs found in organisms often exhibit minimal amino acid mutations, and such small differences in peptide conformation may be utilized to design more selective AMPs.

The effect of phosphatidylcholine depletion on biochemical and physical properties of a Neurospora crassa membrane mutant.

By using the choline starvation process it is possible to deplete the membranes of Neurospora crassa choline auxotroph chol-1 of phosphatidylcholine, without affecting the viability of germinated spores or whole mycelium. Spin label probes were used to examine the possible dependence of the physical state of cellular lipids on the presence of phosphatidylcholine in the membranes. Increased freedom of rotational motion of lipid soluble probes was regularly detected in choline-starved mycelium. The accumulation of neutral lipids (mostly triglycerides) in bulk form was also observed during the choline starvation process. The experiments with isolated and separated lipid classes indicated that the observed increase in fluidity of lipids in choline-starved mycelium is partly due to the difference in physical properties between bulk lipids and membrane lipids. Spin label probe 2N4 (2-propyl-2,5,5-trimethyloxazolidine-N-oxyl), which can partition at the membrane-water interface, exhibited easier partitioning among membrane lipids of choline-starved mycelium.

Secondary structure predictions and medium range interactions.

Several authors have proposed that predictions of protein secondary structure derived from statistical information about the known structures can be improved when information about neighboring residues participating in short and medium range interactions is included. A substantial improvement shown here indicates that current methods of including this information are not more successful than methods that do not. Evaluations of the Chou and Fasman method (Adv. Enzymol. 47 (1978) 45-148), that does not include information about interactions (except in averaging), have shown it to be about 49% correct for three states (helix, beta-sheet and undefined). In comparison, the method of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120), that explicitly includes information about neighboring residues, has an accuracy of 57% residues correct for three states. However, we have obtained an 8% improvement for predictions of secondary structure based on the algorithm by Chou and Fasman. The improvements are obtained by eliminating many rules and by choosing the best decision constants for structure assignments. The simplified method described here is 57% correct for three states using preference values calculated in 1978.

Interactions between a new class of eukaryotic antimicrobial agents and isolated rat liver mitochondria.

Members of a newly discovered class of eukaryotic antimicrobial peptides are shown to release respiratory control in isolated rat-liver mitochondria. They also dissipate the membrane potential and inhibit respiration. The uncoupling activity of the peptides decreases with time probably due to the presence of proteases in the mitochondrial preparation. Quinine and Mg2+ reduce the activity of the peptides. The nature of the dependence of the respiratory rate on the concentration of added peptides suggests that they are active in a multimeric form, consistent with the formation of a channel across the inner mitochondrial membrane. The channel allows passage of sucrose.

Serum paraoxonase activity in dairy cows during pregnancy.

Preparturient dairy cows are at high risk of metabolic and reproductive disorders and oxidative stress is considered to be involved in these events. We investigated the serum paraoxonase activity in dairy cows during pregnancy and alterations in lipid and lipoprotein patterns in this period. The relation between paraoxonase activity and HDL-cholesterol concentration was also compared. The study was carried out on 76 pregnant lactating and 26 pregnant dry Holstein dairy cows. The serum paraoxonase activity was determined by the method of hydrolysing of paraoxon, while triglyceride, cholesterol and HDL-cholesterol concentrations were measured by the enzymatic kit methods. A significantly higher serum triglyceride concentration (P<0.001) was observed in dry cows compared to lactating cows. The total cholesterol and HDL-cholesterol concentrations were significantly lower (P<0.001) in dry cows than in lactating ones. In dry cows, paraoxonase activity was significantly lower than in those lactating (P<0.001). There was no significant difference in paraoxonase/HDL-cholesterol ratio between the investigated groups. It seems that the lower HDL concentration could be one of the causes of reduced paraoxonase activity considering the role of HDL as a carrier of most paraoxonase molecules in the blood. A decreased serum paraoxonase activity could diminish the effectiveness and total capacity of the whole antioxidative system during prepartum period in dairy cattle.

Magainin 2 amide and analogues. Antimicrobial activity, membrane depolarization and susceptibility to proteolysis.

We compared the abilities of synthetic magainin 2 amide and its analogues to inhibit the growth of Escherichia coli and to cause membrane depolarization in E. coli cells and cytochrome oxidase liposomes. The analogue, magainin A, was about 40-times more active than magainin 2 amide in inhibiting the growth of E. coli and had a much more sustained effect on the membrane potential. In the liposomal system, however, there was only approx. 20% difference between these two peptides in the reduction of membrane potential and uncoupling of respiration. Studies with pronase digestion suggested that the difference in potency may be due to differential susceptibility to proteolysis in the presence of membranes.

A liver urocanase deficiency.

Two sisters with a rare inborn error of histidine metabolism resulting from urocanase deficiency are being presented. The more common form of familial histidinemia due to histidase deficiency is excluded. The urocanase deficiency is proven by demonstrating increased excretion of metabolites of the product of the urocanase enzyme action. Further, the strongest evidence for the urocanase defect rests on the demonstration of urocanase deficiency and normal histidase activity in liver.

Follicular fluid contents of hyaluronic acid, follicle-stimulating hormone and steroids relative to the success of in vitro fertilization of human oocytes.

OBJECTIVES: To determine the concentrations of hyaluronic acid, FSH, P, and E2 in the follicular fluid (FF) obtained from IVF-ET patients and to assess the value of these measurements in predicting the outcome of fertilization. DESIGN: One hundred eleven samples were retrospectively analyzed for the hyaluronic acid and hormone contents. SETTING: University-based tertiary care center. PATIENTS: Preovulatory FF samples were collected from 67 women undergoing IVF-ET treatment because of tubal absence or obstruction. MAIN OUTCOME MEASURES: The FF hyaluronic acid and hormone concentrations were compared according to the type of ovulation induction, follicular development, and IVF outcome. RESULTS: According to the type of ovulation induction, a significantly lower hyaluronic acid concentration was found in FF harvested from the patients treated with GnRH agonist-hMG. No significant correlation was found between FF hyaluronic acid and either morphological maturity of the oocyte-cumulus complex or fertilizability of oocytes. The level of FSH was significantly higher in FF, yielding a mature oocyte-cumulus complex and from which the oocyte obtained successfully fertilized and cleaved. A significant increase in the E2 concentration was found in FF in which mature cumuli oophori were present. The levels of hyaluronic acid significantly correlated with FSH in FF. CONCLUSIONS: Expansion of the human oocyte-cumulus cell complex is an FSH-dependent phenomenon. The data are in agreement with the hypothesis that intrafollicular FSH plays an important role in the secretion of hyaluronic acid by granulosa cells and may act synergistically with E2 to enhance cytoplasmic maturation, resulting in successful fertilization.

Variation of efficiency with free-energy dissipation in models of biological energy transduction.

For two models of biological free-energy transducers, it is investigated how free-energy dissipation and efficiency vary as (i) the demand for output free energy, (ii) the input free energy or (iii) the properties of the transducers themselves, are varied. One model is representative of near-equilibrium free-energy transducers in general, the other is a special case of far-from-equilibrium free-energy transduction, reminiscent of proton pumping by bacteriorhodopsin. It turns out that the relationship between efficiency and free-energy dissipation depends strongly on what varies. In some cases, free-energy dissipation increases as the efficiency increases. It is suggested that this is one reason why biological evolution has not resulted in high efficiencies and low rates of free-energy dissipation. For the near-equilibrium free-energy transducer, the free-energy dissipation at the static head steady state is minimal with respect to variations in the output force. For the far-from-equilibrium model (of bacteriorhodopsin), the static head does not correspond to such a minimum, if that free-energy transducer slips.

Altered distribution of urinary glycosaminoglycans in diabetic subjects.

We studied the influence of type 1 and type 2 diabetes mellitus with similar duration on the urinary excretion of total glycosaminoglycans and alteration of urinary glycosaminoglycan distribution pattern. Investigations were performed in the 24-hour urine samples of 31 children with type 1 diabetes mellitus, 36 adults with type 2 diabetes mellitus and in 30 age-matched controls for each group. We found that type 2 diabetes mellitus also induced an increased urinary excretion of total glycosaminoglycans and that both type 1 and type 2 diabetes alter the urinary distribution of heparan sulphate and dermatan sulphate. Observed changes correlate with duration of the disease. Microalbuminuria was detected in 9 of 36 type 2 adult diabetics (25%). The microalbuminic group had a significantly higher heparan sulphate and/or dermatan sulphate excretion rate. To clarify whether an altered urinary distribution of heparan sulphate and dermatan sulphate may precede the development of microalbuminuria, it is necessary to performed a prospective study in which urinary glycosaminoglycans and microalbuminuria are measured year by year starting from the diagnosis of diabetes.

Effect of 'antidiabetis' herbal preparation on serum glucose and fructosamine in NOD mice.

The antihyperglycemic effect of the Antidiabetis herbal preparation ((Myrtilli folium (Vaccinium myrtillus L.), Taraxaci radix (Taraxacum officinale Web.), Cichorii radix (Cichorium intybus L.), Juniperi fructus (Juniperus communis L.), Centaurii herba (Centaurium umbellatum Gilib.), Phaseoli pericarpium (Phaseolus vulgaris), Millefollii herba (Achillea millefolium L.), Morii folium (Morus nigra L.), Valeriane radix (Valleriana officinalis L.), Urticae herba et radix (Urtica dioica L.)), patent No. P-9801091 Zagreb, Croatia was investigated. Two extracts were prepared: ethanol extract (extract 1), and ethanol extract from which ethanol was evaporated on a rotatory evaporator at a temperature of 45 degrees C (extract 2). Extract 1 and extract 2 were administered (in experiment 1) to alloxan-induced non-obese diabetic (NOD) mice in the same dose of 20 mg/kg. Blood glucose was determined before, and 10, 30, 60 and 120 min after the preparation administration. Extract 1 and extract 2 decreased the level of blood glucose by 10 and 20%, respectively, of the initial value (at 0 min, mean = 22.6 +/- 8.3 mmol/l). Serum levels of glucose and fructosamine were determined in NOD mice, NOD mice administered extract 2 in a dose of 20 mg/kg of extract 2, and NOD mice administered acarbose in a dose of 25 mg/100 g chow, in order to verify the hypoglycemic action of extract 2 (in experiment 2). Extract 2 and acarbose were admixed to the chow. The duration of treatment was 7 days. Significantly lower glucose (P < 0.05) and fructosamine (P < 0.001) levels were recorded in extract 2 treated NOD mice as compared with NOD mice. Study results showed extract 2 to significantly decrease the level of glucose and fructosamine in alloxan induced NOD mice. Our future studies will be focused on the search of active principles of the extracts.

Serum paraoxonase activity and lipid parameters in the early postpartum period of dairy cows.

The effect of early lactation on serum paraoxonase activity was studied on 21 postpartum dairy cows and 19 non-pregnant late lactating dairy cows. A significant decrease of the paraoxonase activity was found in the early postpartum period compared to the late non-pregnant lactation. The serum triglyceride, cholesterol and LDL-cholesterol concentration were also markedly reduced during the postpartum period, while the serum HDL-cholesterol concentration showed no significant change. The results indicate that lower serum paraoxonase activity is associated with lipid metabolic disorders in the early postpartum period. A decreased serum paraoxonase activity may lead to the reduction of the antioxidative capacity and antioxidative protection during the early postpartum period.

Lipid status, paraoxonase-1 activity and metabolic parameters in serum of heifers and lactating cows related to oxidative stress.

The objective of this study was to investigate serum lipids, metabolic parameters and activity of the anti-oxidative enzyme paraoxonase-1 (PON1). The study was conducted on non-pregnant heifers with optimal health status and on healthy dairy cows in the period of intensive lactation, assuming that the energy and metabolic demands during lactation reduce anti-oxidative protection. Total cholesterol and HDL-cholesterol concentrations were significantly higher (P<0.05) in lactating cows than in heifers. Bilirubin concentration and γ-glutamyltransferase (GGT) activity were also significantly higher in lactating cows (P<0.05), indicating increased hepatic efforts of cows to meet energy requirements for lactation. Significantly lower PON1 activity and PON1/HDL ratio in lactating cows compared to heifers (P<0.05) showed that metabolic efforts during pregnancy, parturition and lactation influence PON1 activity due to oxidative stress. Concurrent increase in total and HDL-cholesterol during lactation indicated that the HDL particle is a major carrier of cholesterol in cows.

Cyanide-resistant respiration of a Neurospora crassa membrane mutant.

Choline starvation of the Neurospora crassa chol-1 mutant leads to a decrease in respiration through the cytochrome chain and a concomitant induction of the alternate oxidase.

Lecithin requirement for the sporulation process in Neurospora crassa.

Reversible inhibition of conidiogenesis occurred when lecithin was depleted from Neurospora membranes by choline starvation.

Effect of ochratoxin A on brush border enzymes of rat kidney.

Ochratoxin A was given orally at 60 microgram/kg body weight in neutral olive oil to Fischer rats for 30 days, at which time they were killed. Clinical state, weights of animals and of their organs and urea and creatinine concentrations were not affected during the exposure period. Significant increases in the activity of enzymes in urine were found: 60% increase in alanine aminopeptidase, 45% increase in gamma-glutamyl-transferase and 90% increase in alkaline phosphatase. These changes indicate early pathological changes in the kidney. Relatively small amounts of the toxin thus affect kidney membrane cells.

Current system of undergraduate and postgraduate education in clinical chemistry in Croatia.

We present here a specific model of education and practice in clinical chemistry that is almost exclusively based on the medical biochemists academically educated at the Faculty of Pharmacy and Biochemistry. This model has been successfully used for 35 years in Croatian Health Care System. Undergraduate education in clinical chemistry consists of four years of specific university education which provides for all requirements to maintain the high quality of our profession. Postgraduate education leading to more specific scientific and professional expertise is further regulated by the laws issued by The Ministry of Health and The Ministry of Science and Technology. At present there is a compulsory programme of lifelong continuing education recognised by Croatian Chamber of Medical Biochemists.

Application of the monodimensional electrophoresis for the separation of glycosaminoglycans on Cellogel cellulose acetate strip.

A monodimensional electrophoretic method for the separation of glycosaminoglycans on Titan III Zip Zone cellulose acetate plate based on their different electrophoretic mobilities in barium acetate and different solubilities in ethanol was applied to the Chemetron electrophoretic equipment. Improved timing of individual steps of electrophoretic run, additional cooling and pressure must be introduced for optimal separation of glycosaminoglycans mixture (dermatan sulphate, heparan sulphate, hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate and keratan sulphate) resulting in five well separated sharp bands. By all these changes in the original procedure of Hopwood and Harrison, the separation of chondroitin-4-sulphate and chondroitin-6-sulphate was not achieved. The modified procedure on Cellogel strip is suitable for the screening of mucopolysaccharidoses.